1. Field of the Invention
This invention relates to the field of immunotherapy, and more particularly immunotherapy with low doses of cytokines.
The present method and products are suitable for the treatment of diseases or conditions such as microbial infections, cancer and the like.
2. Description of the Background
In the immune system, there are three major types of lymphocytes: B cells, T cells, and natural killer (NK) cells. B-cells are derived from bone marrow, and comprise about 10% of the lymphocytes found circulating in blood. When stimulated by a specific antigen, each B-cell differentiates into a plasma cell that secretes antibodies of a single specificity. T-cells mature in the thymus and, make up about 80% of circulating lymphocytes. Although not producing antibodies, T-cells bear on their surfaces specific antigen receptors resembling antibody molecules. T-cells react to antigen stimulation by secreting immunomediator molecules or cytokines (helper T-cells), and toxic molecules (cytotoxic T-cells). Cytotoxic T-cells act directly on infected cells, and by secreting toxic molecules kill them and any foreign particles, such as microorganisms, they may contain. NK cells, make up about 10% of the lymphocyte population, and are not antigen specific, but recognize and kill cells infected with microbes. Monocytes and macrophages are large scavenger cells that ingest foreign particles and present antigens to the T-cells, which trigger specific immune responses. When an antigen is introduced, it is initially ingested by macrophages and other antigen presenting cells. After digestion, short segments thereof are presented on their cell surfaces. Only a few of all circulating T-cells have receptors that specifically bind to the antigen, and this binding stimulates the T-cells to secrete cytokines.
Cytokines are small proteins secreted primarily, but not exclusively, by cells of the immune system that promote the proliferation and/or differentiative functions of other cells. Examples of cytokines include interleukins, interferons, hematopoietic colony stimulating factors (CSF), and proinflammatory factors such as tumor necrosis factor (TNF). The therapeutic stimulation of the immune system has yet to find broad applications because of the difficulty in avoiding toxicity, which is part and parcel of immune-mediated inflammation. The toxicity associated with immunoreactivity is familiar to everyone as the signs and symptoms that occur during microbial infections, such as fever, fatigue, malaise and myalgia. These toxic symptoms were originally thought to be caused by substances, such as toxins, released or produced by the microbes themselves. Within the past decade, however, it has gradually become appreciated that the toxic signs and symptoms associated with microbial infection are attributable to molecules termed cytokines released, upon activation, by the immune system. When stimulated, for example, by microbes, the immune system produces the cytokines, which themselves, i.e. not the microbial toxins, produce the recognizable unpleasant and harmful effects. This was proven when the cytokines themselves were isolated, purified and injected into experiential animals and humans.
Despite producing toxic symptoms, the cytokines are primarily responsible for regulating the immune system. They determine the onset, magnitude, and duration of the immune response by stimulating the proliferation and differentiation of various types of cells comprising the immune system, including all of the white blood cells (leukocytes) that are recognizable as lymphocytes, monocytes/macrophages, polymorphonuclear leukocytes (PMN), and specialized antigen-presenting cells (APCs). When it was realized that cytokines regulate the immune system, it was hoped that they could be used therapeutically, to boost or augment immune reactivity in the treatment of microbial infections, malignancies, and for various immunodeficient states, such as the Acquired Immune Deficiency Syndrome (AIDS). However, the high and sometimes intolerable toxicities associated with cytokine administration have precluded their widespread use, especially in asymptomatic individuals afflicted with an infection or illness without signs or symptoms, such as infection with the Human Immunodeficiency Virus (HIV).
The toxicities associated with the administration of various cytokines at high doses are severe. For example, one of the first cytokines to be discovered, interleukin 2 (IL-2), was used initially to treat cancer in very high doses, up to 150xc3x97106 IU/day (10 mg/day). This resulted in extremely toxic side effects, including capillary leak with hypotension and high fever ( greater than 39xc2x0 C.). This limited the duration of IL-2 therapy to only a few days, and restricted its use to solely patients hospitalized in the intensive care unit.
Over the past several years, lower doses of cytokines have been administered in attempts to circumvent most of the toxicities, while hoping to still retain at least some of the immune enhancing effects. For example, Caliguri and co-workers, including the inventor, found that ambulatory cancer patients could tolerate IL-2 administered as a continuous intravenous (IV) infusion for up to 90 days with minimal toxicities (WHO Grade 1, see table) provided the dose was in the range of 2.5-5.0xc3x97105 IU/m2 body surface area per day. Most importantly, even though the dose was lowered about 600-fold from the original dose used earlier in the treatment of cancer, there were still detectable augmenting effects on the immune system, notably a gradual increase in the concentration of circulating Natural Killer (NK) cells. Similar results were recently reported by Bernstein and co-workers, who administered IL-2 subcutaneously (s.q.) to patients suffering from AIDS-associated malignancies, in doses ranging from 0.5-1.0xc3x97106 IU for 3 months. In a separate study, Teppler and co-workers, including the present inventor, injected IL-2 daily for 30 days intradermally (i.d.) into asymptomatic HIV+ individuals. At a dose of 0.18xc3x97106 IU/day given for this short interval, no untoward toxic effects were noted, but neither was there an increase in circulating leukocytes.
Thus far, the following four (4) families of cytokines that regulate the immune system, are recognizable according to their structures.
1) The interleukin family includes cytokines such as IL-2, 3, 4, 5, 6, 7, 9, 12, 13, and 15. These cytokines are small (10-20 kDaltons) proteins that all share a 3 dimensional structure of 4 antiparallel alpha helices. The receptors of this cytokine family share amino acid sequence homologies, especially in their extracellular domains.
2) The tumor necrosis factor (TNF) family includes compounds such as TNF-xcex1, TNF-xcex2 (lymphotoxin), nerve growth factor (NGF), and the CD40, Fas, CD27, and CD30 ligands. The ligands of this family are either secreted or remain membrane anchored, and function as homotrimers of about 15 kDalton monomers. The receptors of this family share amino acid sequence homologies.
3) The interferon (IFN) family includes compounds such as IFN-xcex1, IFN-xcex2, and IFN-xcex3, and is distinguished by the unique biologic property of stimulating cells to prevent viral replication.
4) The chemokine family includes molecules such as IL-8, macrophage inhibiting protein (MIP), and Rantes. These cytokines are small (about 10 kDaltons), and bind to a distinct family of receptors that have 7 membrane spanning alpha helices, and that are coupled to guanine nucleotide binding proteins (G proteins).
Even though these distinct cytokine families may be classified based upon their structure, they have in common the properties of being produced in, and/or being active on, the mammalian immune system.
Interleukin 2 (IL-2), for example, was one of the first cytokines to be identified and characterized. It is produced exclusively by T-lymphocytes in response to stimulation by antigens. IL-2 acts on the three major types of lymphocytes, including T cells, B cells, and NK cells, stimulating them to proliferate and augmenting their differentiative functions. IL-2 potentiates both innate or natural host defenses by stimulating NK cells, and antigen-specific acquired immune reactivity by stimulating T cells and B cells.
In vitro studies have shown that IL-2 mediates its effects by binding to specific receptors (IL-2R) expressed on the surfaces of IL-2-responsive target cells. When IL-2 binds to its receptors, it initiates a series of intracellular events that result in the activation of a set of genes, the products of which determine the cellular responses observed, such as cellular proliferation. Thus, one of the consequences of IL-2 stimulation is an expansion in the number of T-cells, B-cells and NK cells. IL-2 also stimulates the expression of genes encoding other cytokines, especially by T-cells and NK cells. In particular, IL-2 stimulation of NK cells results in their production of secondary, pro-inflammatory cytokines, including TNFxcex1, IFN-xcex3 and GM-CSF. These NK cell-derived cytokines, in turn, are potent stimuli for monocytes, promoting their production of further pro-inflammatory cytokines. Thus, although IL-2 is attractive as an immunotherapeutic agent, given that it stimulates all of the major types of lymphocytes, its therapeutic use has been impeded by its toxicity, most of which has been attributed to the release of large quantities of secondary cytokines. Clearly, the major problem associated with the administration of IL-2 has been its toxicity, which has prevented its use in therapy for a wide range of illnesses and indications.
Various studies on IL-2 immunotherapy have been reported, the initial ones dating back to 1984. Since that time, various doses, routes of administration and schedules of administration have been tried. However, up to the present time, IL-2 has only been approved by the Food and Drug Administration for limited use in patients with renal cell carcinoma. For this purpose, the high doses of IL-2 used, only permit its administration for 1-2 weeks before severe toxicity develops. Additional rounds of treatment administered after discontinuance proved ineffective.
Throughout this patent, where possible, all reported cytokine units of biological activity have been converted to International Units (IU) to enable a comparison of the doses of the same cytokine used in different studies. Also, where possible, and when appropriate, cytokines will be referred to as the amount of protein, in weight or moles for uniformity""s sake. In addition, some agents"" doses are expressed as either IU or moles per m2 of body surface area (BSA). The BSA values are easily calculated from similar values based on a subject""s weight and height using a standard conversion table of height and weight measurements. For example, a 70 kg. person of normal height has 1.5 m2 BSA. Doses of 0.15 to 15xc3x97106 IU impure, natural IL-2/m2/day were administered to severely ill AIDS patients by subcutaneous (s.q.) or intravenous (i.v.) injection for a short period (3 weeks) or for 5 days/week for 4 weeks. Neither significant toxicity nor improvements in immunologic parameters were seen. The first administration of recombinant interleukin-2 (rIL-2) was reported in 1985, when cancer patients were given intermitent daily doses of up to 70xc3x97106 IU rIL-2/m2/day for 1-2 weeks, with severe toxicity WHO grades 3 and 4. (See, Table 3 below). In spite of the severe toxicity elicited, this dose continued to be used, with minor variations on dose and schedule, in the past 10 years in the treatment of many malignancies and other indications because of its beneficial anti-tumor effect. The doses used varied from 5xc3x97103 and 12xc3x97106 IU IL-2/m2/day and the time of administration varied from just a few days to 90 days, in some cases the administration being done only a few days every week. These studies proved disappointing, mostly due to their lack of efficacy or to the toxicity elicited, which varied from grade 1 to grades 3 and 4 in some cases, or both. U.S. Pat. No. 5,026,687 discloses the use of ddI for the treatment of HIV+ patients. Example 8 is a paper example forecasting the oral administration of ddI in combination with an all encompassing dose of IL-2 (25,000-1,000,000 U/day) by continuous infusion or other systemic administration for a period of 3 months. Although the example indicates that xe2x80x9cbeneficial results are seenxe2x80x9d, because of its hypothetical nature, it provides no real information on IL-2 toxicity since no enabling work was provided. In summary, immunotherapy with high doses (greater than 1xc3x97106 IU/m2/day) of IL-2 has been shown to lead to severe, unacceptable toxicity WHO grades 2 and higher, requiring hospitalization. These doses are only tolerable for a few days, and result in only transient detectable improvements in immune function. Lower doses (0.1-0.5xc3x97106 IU rIL-2/m2/day) have been administered for up to 90 days but resulted in toxicity WHO grade 1, with only transient increases in immunological function.
Thus, up until the time of this invention, the view was widely held that the use of cytokines was ineffective in some diseases, and contraindicated, for example, in patients with HIV infection due to its potential for activating or increasing replication of HIV. More generally, up to the present time, no long term cytokine therapy has been proven suitable, effective and safe, for the treatment of HIV infected individuals in the absence of severe side effects. Nor have there been any reports on the administration of cytokines to immune impaired infants or mammalian animals, or to normal individuals, infants or mammalian animals which may be temporarily afflicted with an infection or other condition, and who would benefit from a temporary stimulation of their immune system to overcome the condition.
Accordingly, up to the present invention, it was not believed possible to administer cytokines, whether in a preventative or therapeutic mode, for prolonged periods of time, so that individuals could carry on normal lives and experience improved immune function, without eliciting toxicity. Moreover, up to the present time, cytokines had never been administered to asymptomatic individuals for longer than 30 days, so that the long-term effects of its chronic administration remained unknown.
Thus, there is still a need for a maintenance method of administering agents having cytokine activity at a dose which stimulates the immune system, while producing minimal or no toxicity and/or detrimental side effects which have precluded the continued administration of the same cytokines, alone and/or in combination with other therapeutic agents. Such method would permit the effective avoidance, and/or amelioration of symptoms, which afflict certain subjects with decreased immune response or an impaired immune system, in a safe manner, permitting them to resume an active life schedule.
This invention relates to a unit dosage composition comprising, in a sterile container, an agent having cytokine activity, including natural, recombinant and mutated cytokines, fragments, analogs, and derivatives of the cytokines, and mixtures thereof. The composition is also provided as a kit with single or multiple unit dosages of the composition, instructions, and device(s) for its administration, such as needles and syringes, inhalators, and the like. The composition may be provided in various forms, including topical and systemic forms, such as powders, creams, ointments, sprays, solutions, suppositories, powders, suspensions, patches, emulsions, implants, and encapsulated particles, among others, and may contain other therapeutic agents, such as various forms of the cytokines, including natural, recombinant and mutated forms, fragments, fusion proteins, and other analogues and derivatives of the cytokines, mixtures, other biologically active agents and formulation additives, and mixtures thereof. Topical and controlled release formulations, implants, inhalators, and transdermal, intradermal, transbuccal, and transpulmonary delivery devices, among others, are also included.
The composition of the invention may be self-administered by any topical or systemic route, as long as its peptide bonds are protected from degradation. The present agent is suitable for the chronic stimulation and/or maintenance and/or inhibition of immune response in a subject when administered at a dose effective to activate high affinity cytokine receptors without eliciting substantial toxicity, i.e., WHO group 1 or higher. The amount of the agent administered may be determined as described herein, and is preferably effective to produce about 10 to about 90% or higher saturation of the biologically relevant, high affinity cytokine receptors. The present immunotherapy is useful for stimulating and/or inhibiting the immune system in the prevention and treatment of malignancies, of mild and severe infections afflicting normal individuals, of opportunistic infections generally arising in immunocompromised individuals, such as viral, fungal, parasitic, and bacterial infections, including patients infected with the human immunodeficiency virus (HIV), and infections frequently encountered after operations or procedures, such as bone marrow transplants (BMT), inflammation, necrosis, sepsis, and as an adjuvant for vaccines, among other applications. The present immunotherapy is also generally suitable for the stimulation or inhibition of the immune system in general or of specific immune responses, in normal subjects, children, and the elderly, and for use in animals in general.